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Electronic Supplementary Material (ESI) for Nanoscale Horizons. This journal is © The Royal

Society of Chemistry 2019

Remote-controlled multi-enzyme system for enhanced tumor therapy

via dark/light relay catalysis

Ying Chen,‡ Zi-Hao Li,‡ Jing-Jing Hu, Si-Yuan Peng, Lei Rong,* Yunxia Sun* and Xian-Zheng

Zhang*

Key Laboratory of Biomedical Polymers of Ministry of Education & Department of Chemistry,

Wuhan University, Wuhan 430072, P. R. China

* Corresponding authors. E-mail addresses: rong-lei@outlook.com (L. Rong), yx-

sun@whu.edu.cn (Y. X. Sun), xz-zhang@whu.edu.cn (X. Z. Zhang)

‡ These authors contributed equally to this work.

Electronic Supplementary Material (ESI) for Nanoscale Horizons.This journal is © The Royal Society of Chemistry 2019

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Experimental Section

Materials: Potassium ferricyanide, polyvinylpyrrolidone (PVP K30), poly

(dimethyldiallylammonium chloride) (PDDA) and hydrogen peroxide was obtained from

Sinopharm Chemical Reagent Co. 3,3',5,5'-Tetramethylbenzidine and purified terephthalic acid

were purchased from Sigma Aldrich. 5,5-dimethyl-1-pyrroline-1-oxide and 2,2,6,6-

Tetramethylpiperidine were purchased from Adamas Reagent. The BCA reagents and the

Superoxide Dismutase (SOD) assay kit were purchased from Beyotime. Cy5-NHS and glucose

oxidase were purchased from Aladdin Reagent Co. Ltd. (Shanghai, China). Aminophenyl

fluorescein (APF), Singlet oxygen sensor green (SOSG), 2',7'-Dichlorodihydrofluorescein

diacetate (DCFH-DA), Calcein-AM, and propidium iodide (PI) were obtained from Sigma-

Aldrich. All the reagents used were obtained from commercial suppliers without further

purification.

Preparation of PB, HMPB, GPB and mGPB: PB particles were synthesized according to a

previous report. Briefly, 131.7 mg of K3[Fe(CN)6]·3H2O and 3 g of PVP were dissolved into

40 mL of HCl solution (0.01 M) under strring conditions for 30 min, then heated to 80 °C for

20 h. And the synthetic products were acquired by centrifugation and washed with ethanol and

deionized water for three times.

HMPB was obtain by an acid etching method. 20 mg of PB and 100 mg of PVP were

dissolved into 20 mL of HCl solution (1.0 M). Then the mixture solution was added into a

Teflon vessel and heated at 140 °C for 4 h. After cooling and centrifuging, PDDA was added

to gain HMPB nanocube with positive potential. The obtained HMPB nanoparticles (20 mg)

were dissolved into 10 mL of deionized water, then 2 mg of GOx was added. After 12 h stirring,

the GPB was collected by centrifugation and washed with deionized water for three times. 1

mg of GPB and 0.5 mL of 4T1 cell membrane (2 mg/mL) were dissolved into deionized water.

Then, the mixture was extruded to obtain mGPB.

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Peroxidase-like activity of GPB: RhB and TMB were chosen as indicators to evaluate the

peroxidase-like activity of GPB. 20 μL of GPB (1 mg/mL) and 10 μL of RhB (9 mg/mL) were

applied to the reaction system. 10 μL of H2O2 (30% w/w) or D-glucose (1 mM) was added as

substrates. DI water was used to fill volume of 1 ml. When excited at 350 nm, the emission at

550 nm of RhB was recorded after 15 min treatment (NIR 660 nm: 30 mW cm−2). TMB, which

can be oxidized to ox-TMB with an absorption of 652 nm was used to further evaluate the

Michaelis–Menten kinetics of POD-like activity for GPB. 0.8 mM of TMB was applied to 20

μL/mL of GPB solution, followed addition of a series H2O2 or D-glucose concentration. All

reactions were monitored by measuring the absorbance of 652 nm at different times. The

Michaelis–Menten constant (KM) and maximal velocity Vmax were calculated according to the

Michaelis–Menten saturation curve.

Oxidase-like activity of GPB: The oxidase-like activity of GPB was evaluated by monitoring

the absorption spectra of ox-TMB at 652 nm. First, O2 was purged in HAC-NaAC buffer

solution (20 mM pH = 5.2) to saturate dissolved oxygen. 1 mL of TMB (3.2 mM) was applied

to 20 μL/mL of GPB solution and HAc-NaAc buffer solution (20 mM pH = 5.2) was used to

fill volume of 3 mL. All reactions were monitored by measuring the absorbance of 652 nm after

15 min treatment (NIR 660 nm: 30 mW cm−2).

Detection of •OH and 1O2: The detection of •OH was first detected based on the reaction of

between terephthalic acid (PTA) and •OH. Upon capturing •OH, negligibly fluorescent PTA

would generate 2-hydroxy terephthalic acid with unique fluorescence around 435 nm. DMPO,

as a trapping probe, were further employed to demonstrate the existence of •OH by ESR. GPB

(1 mg/mL, 5 μL), PBS (pH 7.4, 80 μL), H2O2 (1%, w/w, 5 μL) and DMPO (0.8 M, 10 μL) were

mixed to prepare the test solution. After NIR 660 nm treatment (30 mW cm−2, 15 min), ESR

spectra were recorded. The following instrument settings were used for collecting ESR spectra:

1 G field modulation, 150 G scan range.

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The detection of 1O2 was first identified by Singlet Oxygen Sensor Green (SOSG) (Ex = 504

nm, EM = 525 nm). TEMP, as a trapping probe of 1O2, were further employed to demonstrate

the existence of 1O2 by ESR. GPB (1 mg/mL, 5 μL), PBS (pH = 7.4, 80 μL), H2O2 (1%, w/w,

5 μL) and TEMP (0.8 M, 10 μL) were mixed to prepare the test solution. After NIR 660 nm

treatment, ESR spectra were recorded. The following instrument settings were used for

collecting ESR spectra: 1 G field modulation, 150 G scan range.

Study the immune evasive and tumor-targeted capabilities of mGPB: RAW 264.7 cells were

seeded in the glass bottom dishes at a density of 1×105 cells per well for 24 h. Then the cells

were incubated with Cy5-GPB or Cy5-mGPB for 4 h. After being washed by PBS, the cells

were incubated with Hoechst 33342 at 37 ℃ for 15 min to stain the cellular nuclei. To assess

the tumor-targeting capability of Cy5-mGPB, COS7, CT26, HeLa and 4T1 cells were seeded

onto in the glass bottom dishes at a density of 1×105 cells respectively. After cultured for 24 h,

the cells were incubated with mGPB for 4 h. Afterward, the cells were washed with PBS and

digested by trypsin and incubated with Hoechst 33342 at 37 ℃ for 15 min to stain the cellular

nuclei. Finally, all the cells were visualized by CLSM (Leica TCS SP8).

In vitro cytotoxicity study: The cytotoxicity of mPB and mGPB under different

concentrations was estimated in 4T1 cells by MTT assay. 4T1 cells (6000/well) were seeded in

the 96-well plates for 24 h. The cells were co-incubated with mPB or mGPB for 4 h and

irradiated by 660 nm laser(30 mW cm−2, 6 min). After co-incubation for 24 h, 20 μL of MTT

(5 mg/mL) was added and incubated for another 4 h. Next, the medium was replaced with 150

μL DMSO in each well. Finally, the optical density at 570 nm was measured by a microplate

reader (Bio-Rad, Model 550, USA). The relative cell viability was calculated according to the

following equation: cell viability = (OD570(samples)/OD570(control))×100%, where OD570(samples) was

obtained in the presence of nanoparticles and OD570(control) was obtained without treatment.

Animal tumor xenograft models: All animal experiments were performed according to the

guidelines for laboratory animals established by the Wuhan University Center for Animal

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Center Experiment/A3-Lab. All study protocols were approved by the Institutional Animal

Care and Use Committee (IACUC) of the Animal Experiment Center of Wuhan University

(Wuhan, China). Injecting 4T1 cells or CT26 cells (1 × 106) subcutaneously on the right back

the BALB/C mice (4-5 weeks old) to establish tumor model.

In vivo fluorescence imaging and biodistribution: When tumors reached a size of

approximately 150 mm3 in volume, 200 μL of DiR-mGPB (containing 200 μg/mL mGPB) was

injected into the 4T1 or CT26 bearing mice via the tail vein. Then the mice were anesthetized

and imaged by the IVIS Spectrum (PerkinElmer) at 0 h, 12 h, 24 h, 36 h, 48 h, 60 h, 72 h, 84 h

and 96 h after injection. The mice were sacrificed at 96 h after systemic injection and the major

organs (heart, liver, spleen, lung and kidney) and tumor tissues were harvested for imaging.

In vivo anti-tumor study and histochemistry analysis: When the tumor volume reached 100

mm3, 4T1 tumor-bearing mice were randomly divided into 8 groups (n = 6) and injected via the

tail vein with (1) PBS, (2) HMPB, (3) GPB, (4) mGPB and (5) PBS + Light, 6) HMPB + Light,

7) GPB + Light, 8) mGPB + Light. The light groups were irradiated by 660 nm laser (150 mW

cm−2) for 6 min after injection for 24 h. And tumor volumes and body weights of mice in eight

groups were measured every day. And tumor volume was calculated as tumor length×tumor

width2/2. All the mice were excised after 14 days treatment. Simultaneously, the main organs

(heart, liver, spleen, lung and kidney) and tumors were harvested and used for H&E staining,

immunofluorescence staining, and immunohistochemical staining.

Statistical Analysis: Without specifically mention, all data were averaged from four

independent experiments. Statistical analysis between two groups was performed using a

Student’s t-test, while more than 2 groups were performed using the Two-way ANOVA. The

differences were considered to be statistically significant for a P value < 0.05 (*P < 0.05, **P

< 0.01, ***P < 0.001).

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Table S1. Fitting Results of Fe Element on the Surface of GPB.

Element (%) C1s O1s N1s Fe2p

GPB+H2O2 49.64 11.8 30.95 7.61

GPB+H2O2+NIR 57.87 12.76 24.4 4.97

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Table S2. Room temperature 57Fe Mössbauer parameters of GPB.

System Component IS (mm/s) QS (mm/s) Г/2 (mm/s) Area (%)

LS FeII/III -0.13 0.19 56.9GPB+H2O2

HS FeIII 0.44 0.36 0.21 43.1

LS FeII/III -0.15 0.17 44.6GPB+H2O2+NIR

HS FeIII 0.39 0.42 0.24 55.4

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Table S3. Blood Routine Examination of mice after different treatments with PBS, HMPB,

GPB and mGPB.

Indicator PBS HMPB GPB mGPB

WBC (109/L) 6.24±0.30 6.17±0.37 6.27±0.27 5.67±0.34

RBC (1012/L) 9.32±0.75 9.55±0.52 8.64±0.41 9.19±1.13

HGB (g/L) 125±6.68 127±8.48 116±2.49 125.33±9.81

MCV(fL) 52.56±1.10 52.77±0.65 51.83±0.74 51.40±0.91

MCH (pg) 13.47±0.45 13.33±0.20 13.37±0.32 13.80±0.75

MCHC (g/L) 256.00±2.94 252.33±2.05 258.33±4.49 268.33±1.69

HCT (%) 48.93±3.14 50.47±3.43 44.77±1.82 48.76±5.14

PLT (109/L) 524.33±9.23 511.00±10.82 488.33±15.19 517.33±6.55

MPV (fL) 6.83±0.47 7.26±0.28 6.79±0.22 7.13±0.21

PDW (%CV) 8.03±0.53 9.13±0.87 8.03±0.48 9.27±0.54

PCT (%) 0.23±0.063 0.37±0.024 0.27±0.015 0.26±0.040

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Fig. S1 SEM images of HMPB. Scale bar = 200 nm.

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Fig. S2 SEM images of HMPB. Scale bar = 200 nm.

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Fig. S3 (A) Brunauer-Emmett-Teller (BET) nitrogen adsorption/desorption isotherms and (B)

pore size distribution for GPB calculated by Barrett Joyner-Halenda (BJH) method.

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Fig.S4 Fourier transform diffraction pattern of GPB nanoparticles.

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Fig. S5 The standard curve of BSA for BCA Protein Assay Kit.

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Fig. S6 (A) Fluorescence spectra of RhB degradation. (B) Relative quantification of

absorption intensity.

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0 2 4 6 8 10 12 14 1630

35

40

45

50

55

Tem

pera

ture

(℃)

Time (min)

PBS 2.5 mM 5 mM 10 mM 20 mM

Fig. S7 Temperature curve of GPB under 660 nm (150 mW cm−2).

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Fig. S8 OXD-like activity of GPB.

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Fig. S9 PXRD patterns of GPB with and without irradiation.

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Fig. S10 XPS survey of GPB, GPB +H2O2 and GPB +H2O2+Light.

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Fig. S11 Fourier transform infrared spectroscopy of HMPB, GPB and mGPB.

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400 500 600 700 800 900 1000

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

Abs

orpt

ion

Wavelength (nm)

Fig. S12 UV-vis spectrum of mGPB.

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10 20 30 40 50 60 70 80

Inte

nsity

(a.u

.)

2 (degree)

Fig. S13 PXRD pattern of mGPB.

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PBSmPB

mGPB

PBS+Ligh

t

mPB+Ligh

t

mGPB+Ligh

t0.0

5.0x104

1.0x105

1.5x105

2.0x105

MFI

Fig. S14 Flow quantitative analysis 4T1 cells after stain with DCFH-DA.

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Contro

l

DMSO VC0.0

5.0x104

1.0x105

1.5x105

2.0x105

2.5x105

3.0x105

MFI

NaN 3

Fig. S15 Flow quantitative analysis after treated with different inhibitors.

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Fig. S16 Live/dead cell staining after mPB and mGPB treatment with/without irradiation.

Scale bar = 100 μm.

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Fig. S17 Apoptosis assay of 4T1 cells after different treatments.

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Fig. S18 TUNEL staining of tumor tissues after different treatment with PBS, HMPB, GPB

and mGPB for 14 days.

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Fig. S19 Blood biochemistry analysis of mice (A) liver and (B) kidney.

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Fig. S20 H&E staining against the major organs (heart, liver, spleen, lung, and kidney) after

different treatment with PBS, PBS+Light, HMPB, HMPB+Light, GPB, GPB+Light, mGPB

and mGPB+Light for 14 days. H&E staining: 200× magnification.